Proton-Evolved Local-Field Solid-State NMR Studies of Cytochrome b₅ Embedded in Bicelles, Revealing both Structural and Dynamical Information

Abstract

Structural biology of membrane proteins has rapidly evolved into a new frontier of science. Although solving the structure of a membrane protein with atomic-level resolution is still a major challenge, separated local field (SLF) NMR spectroscopy has become an invaluable tool in obtaining structural images of membrane proteins under physiological conditions. Recent studies have demonstrated the use of rotating-frame SLF techniques to accurately measure strong heteronuclear dipolar couplings between directly bonded nuclei. However, in these experiments, all weak dipolar couplings are suppressed. On the other hand, weak heteronuclear dipolar couplings can be measured using laboratory-frame SLF experiments, but only at the expense of spectral resolution for strongly dipolar coupled spins. In the present study, we implemented 2D PELF (proton-evolved local-field) pulse sequences using either COMPOZER-CP (composite zero degree cross-polarization) or WIM (windowless isotropic mixing) for magnetization transfer. These PELF sequences can be used for the measurement of a broad range of heteronuclear dipolar couplings, allowing for a complete mapping of protein dynamics in a lipid bilayer environment. Experimental results from magnetically-aligned bicelles containing uniformly 15 N-labeled cytochrome b 5 are presented and theoretical analyses of the new PELF sequences are reported. Our results suggest that the PELF based experimental approaches will have a profound impact on solid-state NMR spectroscopy of membrane proteins and other membrane-associated molecules in magnetically-aligned bicelles.

A Model of the Membrane-bound Cytochrome b5-Cytochrome P450 Complex from NMR and Mutagenesis Data

“…The two-dimensional spectrum in Fig. 1C exhibits a distinct circular PISA-wheel pattern of resonances between 60 and 100 ppm, which is indicative of an ␣-helical conformation and was assigned to the transmembrane anchor of cytb 5 based on our previous work (29,50). This is in agreement with previous circular dichroism (51) and Fourier transform infrared (52) experiments, which indicated that the transmembrane domain is at least 50% helical.…”

Understanding solution‐state noncovalent interactions between xenobiotics and natural organic matter using 19F/1H heteronuclear saturation transfer difference nuclear magnetic resonance spectroscopy

“…Because of the low sensitivity of these experiments and, as Dixon et al [33] have reported, the reality that only a small number of xenobiotic molecules may actually be associating with the dissolved SOM at any given moment, much higher solute concentrations than are typically found in the environment are required to ensure the presence of a large enough number of interactions for observation. Mechanistic studies on noncovalent interactions using direct NMR methods, such as those routinely employed in molecular biology, often have to employ model systems in which the conditions are optimized for the analytical technique rather than for their direct relevance to the real world [35]. The NMR experiments used here provide unique information about the xenobiotic interactions that cannot be obtained directly through other analytical means, and, although the experiments are conducted under less than ideal conditions, the type of information obtained is invaluable for developing a fuller molecular-level understanding of the processes influencing contaminant fate in the environment.…”

Proton evolved local field solid-state nuclear magnetic resonance using Hadamard encoding: Theory and application to membrane proteins

“…19 Recently, Ramamoorthy and co-workers reported an elegant application of the PELF pulse scheme to analyze the topology and dynamics of single-pass membrane proteins oriented in magnetically aligned lipid bicelles. 18 Parallel studies demonstrated the utility of the PELF pulse sequence to detect 13 C-1 H dipolar couplings of membrane proteins and lipid bilayers. 30 All of these studies concluded that while the R-SLF experiments provide better resolution for DC in rigid solids and motionally restricted domains of membrane proteins, the PELF experiments offer superior resolution and sensitivity for dynamic molecules and mobile regions of membrane proteins.…”

“…10 SLF experiments have been widely used by chemists and biophysicists to characterize the structures of liquid crystals as well as membrane proteins in mechanically or magnetically aligned lipid bilayers. [11][12][13][14][15][16][17][18] Since the introduction of the SLF experiment, several variants have emerged, such as PISEMA, [19][20][21] SAMPI4, 22 HIMSELF, 23 and more recently the sensitivity-enhanced and constant time versions. [24][25][26][27] These experiments are also known as rotating frame SLF (R-SLF) experiments and take advantage of the heteronuclear DC evolution under Hartmann-Hahn matching of RF fields for I and S spins and homonuclear decoupling for the abundant I spins.…”

“…As a result, the acquisition of multiple 2D experiments at different mixing times is needed to cover the entire range for all the DC values. 18,31,32 This approach is very time consuming and cumbersome, especially for the analysis of membrane proteins oriented in lipid membranes. In fact, these samples are notoriously insensitive due to the low protein-tolipid ratio, which is necessary to maintain the biological activity and homogeneous sample preparations.…”